This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The endoplasmic reticulum-related protein degradation (ERAD) pathway, where misfolded proteins in the ER are transported through the retro-translocon, ubiquinated, and degraded by the proteasome, is essential for cell viability. Before proteasome degradation, the N-glycan on the ERAD substrates is processed by the Peptide:N-glycanase (PNGase). We tried to illustrate the function of PNGase by studying its structure. We recently solved the structure of the catalytic domain of mouse PNGase (mPNGase), which shows a similar fold as the yeast protein. In addition to the catalytic domain, PNGase in higher eukaryotes has both N- and C-terminal extensions. Our structural and biochemical studies on the C-terminal domain of mPNGase identified it as a sugar-recognizing motif. However, the function of the N-terminal domain is unknown. The N-terminal domain has no sequence similarity with other proteins and may adopt a novel fold. Recently it was reported that mPNGase directly interacts with P97/VCP, an AAA ATPase family protein. We proposed an escort model in which P97 functions as a protein interacting platform to present misfolded proteins from the ER to mPNGase and ubiquitinating enzymes. Our biochemical data showed that the N-terminal domain of mPNGase binds to the C-terminal region of P97. This is the first time that a protein-interacting function of the mPNGase N-terminal domain has been established. This is very interesting since it was generally believed that P97 interacts with its effector proteins through its N-terminal domain. The structure of full length P97 has been solved, however, the C-terminal region could not be visualized in the structure. To investigate the function of the N-terminal domain of mPNGase and the details of its interaction with P97, crystallographic studies on these proteins and their complexes are being undertaken. We have recently obtained crystals of the N-terminal domain in its apo-state and in complex with a fragment of P97 and expect them to be ready for data collection and structure solution by the time the RapiData course starts.